The insulin-like growth factor receptor (IGF-IR) is a ubiquitous transmembrane tyrosine kinase receptor that is essential for normal fetal and post-natal growth and development. IGF-IR can stimulate cell proliferation, cell differentiation, changes in cell size, and protect cells from apoptosis. It has also been considered to be quasi-obligatory for cell transformation (reviewed in Adams et al., Cell. Mol. Life Sci. 57:1050-93 (2000); Baserga, Oncogene 19:5574-81 (2000)). The IGF-IR is located on the cell surface of most cell types and serves as the signaling molecule for growth factors IGF-I and IGF-II (collectively termed henceforth IGFs). IGF-IR also binds insulin, albeit at three orders of magnitude lower affinity than it binds to IGFs. IGF-IR is a pre-formed hetero-tetramer containing two alpha and two beta chains covalently linked by disulfide bonds. The receptor subunits are synthesized as part of a single polypeptide chain of 180 kd, which is then proteolytically processed into alpha (130 kd) and beta (95 kd) subunits. The entire alpha chain is extracellular and contains the site for ligand binding. The beta chain possesses the transmembrane domain, the tyrosine kinase domain, and a C-terminal extension that is necessary for cell differentiation and transformation, but is dispensable for mitogen signaling and protection from apoptosis.
IGF-IR is highly similar to the insulin receptor (IR), particularly within the beta chain sequence (70% homology). Because of this homology, recent studies have demonstrated that these receptors can form hybrids containing one IR dimer and one IGF-IR dimer (Pandini et al., Clin. Canc. Res. 5:1935-19 (1999)). The formation of hybrids occurs in both normal and transformed cells and the hybrid content is dependent upon the concentration of the two homodimer receptors (IR and IGF-IR) within the cell. In one study of 39 breast cancer specimens, although both IR and IGF-IR were over-expressed in all tumor samples, hybrid receptor content consistently exceeded the levels of both homo-receptors by approximately 3-fold (Pandini et al., Clin. Canc. Res. 5:1935-44 (1999)). Although hybrid receptors are composed of IR and IGF-IR pairs, the hybrids bind selectively to IGFs, with affinity similar to that of IGF-IR, and only weakly bind insulin (Siddle and Soos, The IGF System. Humana Press. pp. 199-225. 1999). These hybrids therefore can bind IGFs and transduce signals in both normal and transformed cells.
A second IGF receptor, IGF-IIR, or mannose-6-phosphate (M6P) receptor, also binds IGF-II ligand with high affinity, but lacks tyrosine kinase activity (Oates et al., Breast Cancer Res. Treat. 47:269-81 (1998)). Because it results in the degradation of IGF-II, it is considered a sink for IGF-II, antagonizing the growth promoting effects of this ligand. Loss of the IGF-IIR in tumor cells can enhance growth potential through release of its antagonistic effect on the binding of IGF-II with the IGF-IR (Byrd et al., J. Biol. Chem. 274:24408-16 (1999)).
Endocrine expression of IGF-I is regulated primarily by growth hormone and produced in the liver, but recent evidence suggests that many other tissue types are also capable of expressing IGF-I. This ligand is therefore subjected to endocrine and paracrine regulation, as well as autocrine in the case of many types of tumor cells (Yu, H. and Rohan, J., J. Natl. Cancer Inst. 92:1472-89 (2000)).
Six IGF binding proteins (IGFBPs) with specific binding affinities for the IGFs have been identified in serum (Yu, H. and Rohan, J., J. Natl. Cancer Inst. 92:1472-89 (2000)). IGFBPs can either enhance or inhibit the action of IGFs, as determined by the molecular structures of the binding proteins as a result of post-translational modifications. Their primary roles are for transport of IGFs, protection of IGFs from proteolytic degradation, and regulation of the interaction of IGFs with IGF-IR. Only about 1% of serum IGF-I is present as free ligand, the remainder is associated with IGFBPs (Yu, H. and Rohan, J., J. Natl. Cancer Inst. 92:1472-89 (2000)).
Upon binding of ligand (IGFs), the IGF-IR undergoes autophosphorylation at conserved tyrosine residues within the catalytic domain of the beta chain. Subsequent phosphorylation of additional tyrosine residues within the beta chain provides docking sites for the recruitment of downstream molecules critical to the signaling cascade. The principle pathways for transduction of the IGF signal are mitogen activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) (reviewed in Blakesley et al., In: The IGF System. Humana Press. 143-163 (1999)). The MAPK pathway is primarily responsible for the mitogenic signal elicited following IGFs stimulation and PI3K is responsible for the IGF-dependent induction of anti-apoptotic or survival processes.
A key role of IGF-IR signaling is its anti-apoptotic or survival function. Activated IGF-IR signals PI3K and downstream phosphorylation of Akt, or protein kinase B. Akt can effectively block, through phosphorylation, molecules such as BAD, which are essential for the initiation of programmed cell death, and inhibit initiation of apoptosis (Datta et al., Cell 91:231-41 (1997)). Apoptosis is an important cellular mechanism that is critical to normal developmental processes (Oppenheim, Annu. Rev. Neurosci. 14:453-501 (1991)). It is a key mechanism for effecting the elimination of severely damaged cells and reducing the potential persistence of mutagenic lesions that may promote tumorigenesis. To this end, it has been demonstrated that activation of IGFs signaling can promote the formation of spontaneous tumors in a mouse transgenic model (DiGiovanni et al., Cancer Res. 60:1561-70 (2000)). Furthermore, IGF over-expression can rescue cells from chemotherapy induced cell death and may be an important factor in tumor cell drug resistance (Gooch et al., Breast Cancer Res. Treat. 56:1-10 (1999)). Consequently, modulation of the IGF signaling pathway has been shown to increase the sensitivity of tumor cells to chemotherapeutic agents (Benini et al., Clinical Cancer Res. 7:1790-97 (2001)).
A large number of research and clinical studies have implicated the IGF-IR and its ligands (IGFs) in the development, maintenance, and progression of cancer. In tumor cells, over-expression of the receptor, often in concert with over-expression of IGF ligands, leads to potentiation of these signals and, as a result, enhanced cell proliferation and survival. IGF-I and IGF-II have been shown to be strong mitogens for a wide variety of cancer cell lines including prostate (Nickerson et al., Cancer Res. 61:6276-80 (2001); Hellawell et al., Cancer Res. 62:2942-50 (2002)) breast (Gooch et al., Breast Cancer Res. Treat. 56:1-10 (1999)), lung, colon (Hassan and Macaulay, Ann. Oncol. 13:349-56 (2002)), stomach, leukemia, pancreas, brain, myeloma (Ge and Rudikoff, Blood 96:2856-61 (2000), melanoma (All-Ericsson et al., Invest. Opthalmol. Vis. Sci. 43:1-8 (2002)), and ovary (reviewed in: Macaulay, Br. J. Cancer 65:311-20 (1990)) and this effect is mediated through the IGF-IR. High circulating levels of IGF-I in serum have been associated with an increased risk of breast, prostate, and colon cancer (Pollak, Eur. J. Cancer 36:1224-28 (2000)). In a mouse model of colon cancer, increases in circulating IGF-I levels in vivo led to a significant increase in the incidence of tumor growth and metastasis (Wu et al., Cancer Res. 62: 1030-35 (2002)). Constitutive expression of IGF-I in epidermal basal cells of transgenic mice has been shown to promote spontaneous tumor formation (DiGiovanni et al., Cancer Res. 60:1561-1570 (2000; Bol et al., Oncogene 14:1725-1734 (1997)). Over-expression of IGF-II in cell lines and tumors occurs with high frequency and may result from loss of genomic imprinting of the IGF-II gene (Yaginuma et al., Oncology 54:502-7 (1997)). Receptor over-expression has been demonstrated in many diverse human tumor types including lung (Quinn et al., J. Biol. Chem. 271:11477-83 (1996)), breast (Cullen et al., Cancer Res. 50: 48-53 (1990); Peyrat and Bonneterre, Cancer Res. 22:59-67 (1992); Lee and Yee, Biomed. Pharmacother. 49:415-21 (1995)), sarcoma (van Valen et al., J. Cancer Res. Clin. Oncol. 118:269-75 (1992); Scotlandi et al., Cancer Res. 56:4570-74 (1996)), prostate (Nickerson et al., Cancer Res. 61:6276-80 (2001)), and colon (Hassan and Macaulay, Ann. Oncol. 13:349-56 (2002)). In addition, highly metastatic cancer cells have been shown to possess higher expression of IGF-II and IGF-IR than tumor cells that are less prone to metastasize (Guerra et al., Int. J. Cancer 65:812-20 (1996)). A critical role of the IGF-IR in cell proliferation and transformation was demonstrated in experiments of IGF-IR knockout derived mouse embryo fibroblasts. These primary cells grow at significantly reduced rates in culture medium containing 10% serum and fail to transform by a variety of oncogenes including SV40 Large T (Sell et al., Mol. Cell. Biol. 3604-12 (1994)). Recently it was demonstrated that resistance to the drug Herceptin in some forms of breast cancer may be due to activation of IGF-IR signaling in those cancers (Lu et al., J. Natl. Cancer Inst. 93:1852-57 (2001)). Over-expression or activation of IGF-IR may therefore not only be a major determinant in tumorigenicity, but also in tumor cell drug resistance.
Activation of the IGF system has also been implicated in several pathological conditions besides cancer, including acromegaly (Drange and Melmed. In: The IGF System. Humana Press. 699-720 (1999)), retinal neovascularization (Smith et al., Nature Med. 12:1390-95 (1999)), and psoriasis (Wraight et al., Nature Biotech. 18:521-26 (2000)). In the latter study, an antisense oligonucleotide preparation targeting the IGF-IR was effective in significantly inhibiting the hyperproliferation of epidermal cells in human psoriatic skin grafts in a mouse model, suggesting that anti-IGF-IR therapies may be an effective treatment for this chronic disorder.
A variety of strategies have been developed to inhibit the IGF-IR signaling pathway in cells. Antisense oligonucleotides have been effective in vitro and in experimental mouse models, as shown above for psoriasis. In addition, inhibitory peptides targeting the IGF-IR have been generated that possess anti-proliferative activity in vitro and in vivo (Pietrzkowski et al., Cancer Res. 52:6447-51 (1992); Haylor et al., J. Am. Soc. Nephrol. 11:2027-35 (2000)). A synthetic peptide sequence from the C-terminus of IGF-IR has been shown to induce apoptosis and significantly inhibit tumor growth (Reiss et al., J. Cell. Phys. 181:124-35 (1999)). Several dominant-negative mutants of the IGF-IR have also been generated which, upon over-expression in tumor cell lines, compete with wild-type IGF-IR for ligand and effectively inhibit tumor cell growth in vitro and in vivo (Scotlandi et al., Int. J. Cancer 101:11-6 (2002); Seely et al., BMC Cancer 2:15 (2002)). Additionally, a soluble form of the IGF-IR has also been demonstrated to inhibit tumor growth in vivo (D'Ambrosio et al., Cancer Res. 56:4013-20 (1996)). Antibodies directed against the human IGF-IR have also been shown to inhibit tumor cell proliferation in vitro and tumorigenesis in vivo including cell lines derived from breast cancer (Artega and Osborne, Cancer Res. 49:6237-41 (1989)), Ewing's osteosarcoma (Scotlandi et al., Cancer Res. 58:4127-31 (1998)), and melanoma (Furlanetto et al., Cancer Res. 53:2522-26 (1993)). Antibodies are attractive therapeutics chiefly because of they 1) can possess high selectivity for a particular protein antigen, 2) are capable of exhibiting high affinity binding to the antigen, 3) possess long half-lives in vivo, and, since they are natural immune products, should 4) exhibit low in vivo toxicity (Park and Smolen. In: Advances in Protein Chemistry. Academic Press. pp: 360-421 (2001)). Antibodies derived from non-human sources, e.g.: mouse, may, however, effect a directed immune response against the therapeutic antibody, following repeated application, thereby neutralizing the antibody's effectiveness. Fully human antibodies offer the greatest potential for success as human therapeutics since they would likely be less immunogenic than murine or chimeric antibodies in humans, similar to naturally occurring immuno-responsive antibodies. To this end, there is a need to develop high affinity human anti-IGF-IR monoclonal antibodies for therapeutic use.